Malaria remains the most prevalent vector-bone disease of man, estimates of infection ranging from 27 to 488 million persons annually. In the New World tropics, malaria is transmitted by a diversity of Anopheles species which are difficult to separate by standard morphological methods. This project uses an interdisciplinary approach to determine the genetic affinities and evolutionary relationships in the Anopheles nuneztovari sister group which contains known or suspected malaria vectors in most South American countries. New tools for identifying cryptic vector specifies are then coupled with parasite detection and measures of vector population structure at endemic malaria sites in Brasil, Bolivia and/or Ecuador. The results of this project will provide both regional information and general methods for the identification and detection of malaria vectors. New collections will be made in Brasil and Bolivia to characterize species boundaries of Amazonian A. nuneztovari and its sister species A. dunhami and A. rangeli. Genetic divergences and identities will be compared within and among cryptic species through isoenzyme electrophoresis, RFLPs of mtDNA and sequences of selected genes. Larval salivary polytene chromosomes will be studied for inversions and markers, and link-reared progeny of single females will be examined in all life stages. The heritabilities of selected morphological characters will be measured by parent-offspring regressions and through progeny rearings subjected to temperature and density variations in the laboratory. Hypothetical phylogenies will be derived from molecular and morphological data sets. New species will be described, in collaboration with taxonomists, using all available morphological and molecular information. Both biochemical and morphological diagnostic keys will be provided. Species-specific DNA fragments will form the bases of oligonucleotide probes for squash-blot identification of cryptic species. Molecular identifications of cryptic vectors will be coupled at two field sites with parasite detections by sporozoite ELISAs and dissections. A rRNA probe for detecting Plasmodium will be tested. Vector populations will be characterized by insemination, parity, pre-gravid, sporozoite and oocyst rates and measures of body size and larval stress. Relationships between ecological, survival, body size and parasitemia on vector population structure. Progeny of field collections will be sent for testing competency to infection with and transmission of Plasmodium falciparum.